Enteropathogenic Escherichia coli (EPEC) is a frequently isolated diarrheal pathogen in the developing world, and a significant cause of mortality in infants. EPEC attaches to intestinal epithelial cells, subverts their function, and produces the characteristic "attaching and effacing (A/E) lesion". Pathogenesis requires the type III secretion system that directly injects effector proteins into host cells. One of these, EspF, is a 206 amino acid protein with a unique N-terminal sequence followed by three proline-rich 47-amino repeat sequences. EspF is critical for mediating tight junction (TJ) alterations, although it is not required for bacterial viability or A/E lesion formation. Translocated EspF causes a loss in transepithelial resistance, increases monolayer permeability, and redistributes the TJ protein, occludin. In addition EspF also appears to activate the pro-inflammatory transcription factor, NF-kappaB. Our preliminary studies indicate that EspF interacts with various host proteins, including cytokeratin 18 (CK18). Furthermore, host CK18 is redistributed and its interaction with 14-3-3 regulatory proteins is altered following infection with EPEC. 14-3-3 is an abundant, ubiquitously expressed family of proteins that regulate CK18 solubility and distribution, cell-cycle checkpoints, signaling pathways and apoptosis. The long-term goal of this proposal is to determine the mechanism by which EspF mediates host effects. The immediate objectives are to establish the regions or domains of EspF required for interactions, and the corresponding functional significance of the interactions. The domains of EspF required for interaction with CK18, and possibly other host proteins, will be evaluated by using deletion clones of espF in yeast two-hybrid assays and GST pull-down assays. The functional consequences of altering such interactions will be evaluated by assessing epithelial barrier function and inflammation responses following infection with an EspF mutant complemented with the corresponding deletion constructs. The basis of the interaction between CK18 and 14-3-3 will be explored, and the significance of the altered interaction between these two proteins following EPEC infection will be assessed. This proposal will also evaluate the role of 14-3-3 in EPEC infection, assess if EspF directly interacts with 14-3-3, and the effect of 14-3-3 on EPEC induced alterations in barrier function and inflammatory responses.